The long-range goal of the proposed experiments is to understand the function of cardiac myosin binding protein-C (cMyBP-C) in the regulation of myocardial contraction. Mutations in cMyBP-C cause hypertrophic cardiomyopathy and heart failure in millions of people worldwide and under normal conditions cMyBP-C regulates contraction on a beat-to- beat basis. However, neither the mechanisms by which cMyBP-C mutations cause disease nor the mechanisms by which cMyBP-C affects contraction are completely understood. Until now the prevailing hypothesis has been that MyBP-C reversibly limits the speed of contraction by binding to myosin and restricting the ability of myosin heads to extend away from thick filaments and to undergo cycles of interaction with actin on the thin filaments. However, discoveries made by our lab during the first period of this grant challenged this idea and suggest that cMyBP-C itself can bind to actin or other ligands to influence contraction. Aims here will test the role of ligand binding interactions mediated by cMyBP-C by probing the functional significance of novel Molecular Recognition Features (MoRFs) in the regulatory M- domain of cMyBP-C that confer specific binding of the cMyBP-C N-terminus to actin or that mediate other functional effects of the N-terminus in vitro. Understanding the function of the MoRF segment will further provide new insights into disease since HCM missense mutations are clustered within this segment. Specific Aims will 1) map residues in the M-domain MoRFs that mediate actin binding and other functional effects of the M-domain in vitro and in vivo using novel transgenic mice; 2) determine whether the proline-alanine and C1 domains of cMyBP-C outside the M-domain contribute the function of cMyBP-C in vivo using new and existing transgenic mice; and 3) define structural interactions of cMyBP-C with thin and thick filaments. Results from the proposed experiments will provide new insights into the function of MyBP-C and regulatory mechanisms of myocardial contraction in health and disease. PUBLIC HEALTH RELEVANCE: The proposed experiments will investigate the basic molecular mechanisms by which cardiac myosin binding protein-C (cMyBP-C) regulates the strength and speed of heart muscle contraction. Mutations in cMyBP-C are a significant cause hypertrophic cardiomyopathy (HCM), a clinical condition with an incidence of 1 in 500 people. Because HCM is a common cause of sudden cardiac death in adolescents and of heart failure in older populations, improving our understanding of the mechanisms by which cMyBP-C affects cardiac function in both healthy and diseased hearts is expected to ultimately contribute to advancements in disease diagnosis, prognosis, and treatment. Proposed experiments will investigate the ability of cMyBP-C to regulate cardiac contraction by investigating functional effects that result from cMyBP-C binding to thin and thick filaments of muscle sarcomeres.